The studies presented in this proposal will systematically examine the subcellular sites and/or differential rates of intracellular LH and hCG endocytosis and metabolism in vitro, using cultured granulosa cells obtained from preantral follicles of DES treated rats. LH/hCG receptors will be induced with either FSH or 8-Br-cAMP in vitro. Using both iodinated and unlabeled LH and hcG, studies will be carried out to ascertain whether endocytosis and degradation of those gonadotropins occurs through subcellular compartmentalization. Membrane bound and free intracellular LH and hCG will be examined by a variety of techniques including radioimmunoassay, Leydig cell bioassay, Sephadex G-100 and Concanavalin A-Sepharose chromatography, Percoll gradients and electron microscopy using an immunoperoxidase technique with monoclonal antibody to the LH/hCG receptor. The relative contributions of lysosomal and no-lysosomal ATP-dependent proteases on LH and hCG degradation will be systematically examined in cells pretreated with either chloroquine or the metabolic inhibitors 2-deoxyglucose and sodium azide. Choloquine will inhibit lysosomal proteolytic enzymes and the metabolic inhibitors will decrease both lysosomal and the cytosolic proteolytic system; the difference will reflect the contribution of the cytosolic proteolytic system. Monoclonal antibody to the LHhCG receptor will be generated in Balb/c/J mice, using established techniques. That antibody will be used for subcellular localization of receptor to ascertain whether the LH/hCG receptor is predominantly recycled, degraded or a combination of both within gonadotropin responsive cells. A combination of radioimmunoassay and immunoperoxidase techniques with electron microscopy will be used for receptor localization and characterization. The results of experiments presented should provide insights into the specific sites of intracellular LH AND hCG dissociation and degradation as well as ascertain whether LH/hCG receptors are recycled within gonadotropin responsive cells.